PROJECT SUMMARY A major effort in regenerative neuroscience is to improve axon growth after injury to the central nervous system (CNS). Once growth is achieved, however, a second hurdle to improving function is that regenerated axons must succeed in forming synaptic contacts with appropriate sets of post-synaptic neurons. The challenge of restoring effective circuitry is especially acute after spinal injuries that damage the corticospinal tract (CST), a pathway critical for fine motor control. The CST mediates descending motor control by synapsing on specific subsets of spinal neurons, which in humans and rodents alike include a diverse set of interneurons in addition to the direct CST-motor-neuron contacts that characterize primates. The field has achieved increasing success in promoting CST axon growth, yet gains in behavioral recovery have lagged. This work will address the need to monitor the connectivity of regenerated CST axons, and to optimize their behavioral output. To do so we will employ rodent models of spinal injury and capitalize on combined stem cell bridging and viral expression of a pro-regenerative gene called KLF6, which we recently found to evoke robust regenerative CST growth. In addition, we will leverage a recently developed trans-synaptic viral labeling technique that enables an unprecedented ability to visualize post-synaptic target selection. First, we will render KLF6 expression controllable and reversible, in order to silence KLF6 after regeneration occurs in order to determine whether prolonged KLF6 expression itself interferes with behavioral recovery. This will address the pressing question of the degree to which pro-regenerative growth mechanisms may come at the expense of effective synaptic refinement or target selection. Next, we will test the ability of rehabilitative training to sculpt target selection by regenerating CSTs and improve their behavioral output. Finally, we will employ both electrical and chemogenetic means to chronically elevate activity in regenerating CST axons, which we hypothesize will both enhance CST sprouting and improve competition for synaptic territory. These complementary approaches will create optimal strategies to maximize the behavioral benefit that can be extracted from regenerated CST axons.